In recent years, the function of RNA has been greatly expanded from the traditional roles in the protein translation machinery such as messenger RNA and ribosomal RNA to complex regulatory roles such as miRNAs to regulate the expression, splicing and stability of mRNA. Able to accurately profile RNA molecules in a biological sample is necessary to understand the biological system at molecular level and one of the key information to drive the modern systems biology. Two main approaches, hybridization and polymerase chain reaction (PCR) based methods, have been widely used to measure RNA levels; however, these methods are based on prior knowledge on the sequence. Since sequence information is required to generate specific primers (for PCR) and probes (for hybridization) for RNA of interest. These methods have difficulties to accurately measure gene family members with similar sequences. This problem is especially true for a number of noncoding RNA molecules especially miRNA since the sequence length is short, 17 to 25 nucleotide long, and highly similar to each other. Recent development of short high density parallel sequencing technology, Next Generation Sequencing Technology (NextGen), provides a more comprehensive and accurate view of RNA in biological samples. This approach has been used in miRNA profiling as well as to discover new miRNA species; however, the process of preparing sequencing library is tedious and cumbersome which creates problems on the accuracy of measurement. Since miRNA plays important roles in various biological processes, it is necessary to quickly and accurately to measure the level of specific miRNA species.
The nature of miRNA, as compared to short interfering RNA (siRNA), as currently understood is described, for example, in Lee, Y. S., et al., Annu. Rev. Pathol. (2009) 4:199-227. RNA interference with gene expression can be mediated by either siRNA or miRNA. The functional siRNA consists of small double-stranded RNA, while miRNA is single-stranded. The precursor of miRNA is double-stranded and each strand of the miRNA precursor can form functional mature single-stranded miRNA. The mature miRNA is approximately 17-25 nucleotides in length, while siRNA is double-stranded RNA each strand containing about 20-30 nucleotides.
The siRNA that effects gene (or messenger RNA (mRNA)) silencing is formed from cleaving longer double-stranded RNA by Dicer. The sequences of the siRNA correspond to the coding regions of the gene of interest. The short double-stranded synthetic siRNA can also be fed directly to cells to silence the expression of specific gene sequence. The siRNA enters the RNA induced silencing complex (RISC) and represses expression mostly, it is believed, by degradation of targeted gene.
MicroRNA is synthesized in the nucleus initially as pri-miRNA which is then processed to pre-miRNA, a double-stranded RNA molecule with hairpin structure, by the enzyme Drosha. The pre-miRNAs are exported into the cytoplasm by protein called exportin and are then cleaved by Dicer to form a short double-stranded RNA. Usually one of the strands is then incorporated into the RISC to repress translation or the stability of targeted messenger RNAs. The nucleotide sequences in miRNA are generally derived from non-coding portions of the genome including sequences between protein coding genes and introns of protein coding genes. The miRNA usually interacts with the untranslated regions such as the 3′ or 5′ untranslated region of protein encoding mRNA. The actual mechanisms whereby miRNA results in reduction of either protein translation or stability of protein-encoding genes are not completely understood, and appear to differ in various miRNA-mRNA interactions.
A multiplicity of miRNAs is known from many organisms. As of the publication of the above-referenced Lee article, there were more than 5,000 miRNAs from over 50 organisms registered in the database (miRBase release 10.0 August 2007).
The specificity of miRNA is believed to be determined mainly by the first eight nucleotides, labeled the “seed sequence.” Apparently, however, the current algorithms to predict their interacting mRNA targets for the various miRNAs are imperfect. The experimental verification of miRNA interacting targets usually involves fusion of a luciferase reporter to the 3′UTR of the putative target mRNA and showing that light emission is diminished by overexpression of the miRNA.
The terminology applied to microRNA is becoming standardized. It is named as miR plus numbers and miRNAs of similar sequence usually are distinguished by an additional letter following the number—miR-130a and miR-130b will be similar. An additional number is added, e.g., miR-130a-1 if species of the same nature miRNA arise from different genomic loci and have different precursor sequences. The end regions, especially the 3′ end, of miRNAs usual show some length variations. These miRNA sequences, which are derived from the same precursor, are termed isomiR's. This 3′ end sequence heterogeneity may affect the accuracy of miRNA level measurement.
Measurement of particular miRNAs is important in a number of contexts because of the variety of important roles miRNAs play in biological activities, including differentiation, proliferation and cell death. Metabolic activities, such as circadian rhythm and neurotransmitter synthesis also appear to be regulated by miRNAs. Thus the ability of accurately assessing the miRNA spectrum in a biological system is important. However, the nature of miRNA sequence including the very short length of the miRNA sequences as well as highly similar miRNA sequences in the cells makes the measurement problematic, since it makes the designing of primers or probes for specific miRNA difficult. The existence of large number of isomiR's worsens the problem of assessing the level of miRNA accurately.
Several commercially available miRNA measurement platforms are available including microarray based and quantitative polymerase chain reaction (qPCR) based methods. The general detection principles for these methods are well known in the field.
The microarray-based method usually involves the spotting or synthesizing miRNA specific probe sequences on a solid surface, such as glass. The array is then hybridized with florescent or color matrix dye-labeled microRNAs from biological samples. The miRNA levels are then determined by the intensity of fluorescent or colored dyes.
The qPCR based systems generally involve modification of the miRNA templates so that they are long enough to support two independent primers that are required for PCR. In one method, marketed as TaqMan® by Life Technologies, a hairpin nucleic acid molecule with one prong shortened and the other extended to be complementary to the 3′ portion of the targeted miRNA is first annealed to the miRNA in the sample. The longer arm of the hairpin is then further extended by using the miRNA as a template to generate miRNA cDNA. (see FIG. 1). The loop is then opened to serve as an adaptor for extension of the original microRNA thus resulting in a longer RNA molecule for amplification. The amplified molecule is detected with a probe containing a fluorescence quenching system.
In another system, marketed by Qiagen, a polyA tail is added to the miRNA. The miRNA is then transcribed into cDNA by annealing a polyT with a tag sequence to the polyA. The resulting cDNA can then be used in detecting specific miRNA by PCR using an miRNA specific primer and a polyA primer with a tag sequence at 3′end. (See FIG. 2.) The miRNA specific primer usually covers the entire length of the miRNA because of the short sequence of the miRNA.
An additional system, Exiqon, uses a similar approach to that of Qiagen but with different tag sequence. It incorporates modified nucleotides into the primer to provide “locked” nucleic acid, to increase primer specificity.
All of these systems suffer disadvantages, especially because they are dependent on integrity of miRNA sequence at the 3′ end, which is known to be variable within miRNA-isomiR's. The method of the invention offers an improvement that obviates the effect of 3′ end variations by adding adaptors at the 5′ end, which is more conserved.